Figure 4. P4H-TM KO affects passive calcium leak through translocon complex and IP3R. A, B, P4H-TM KO and WT primary cortical astrocytes were loaded with Fluo-4, and live cell calcium imaging was performed in the presence of the indicated pharmacological inhibitors. Representative traces of P4H-TM KO and WT astrocytes are shown on the graphs. Changes in cytosolic calcium [Ca2+]i peak or plateau amplitudes are shown in the diagrams. A, The increase in [Ca2+]i evoked by stimulating the cells with TG (10 μm) was attenuated by the translocon inhibitor anisomycin (200 μm, 10 min before TG), but not by the IP3R inhibitor 2-APB (25 μm, 2 min before TG), n = 22–38 cells per genotype, 5 mice per genotype per condition. Imaging was performed in a calcium-containing buffer. B, Application of an inhibitor of translation (puromycin, PURO, 200 μm) induced a higher increase in [Ca2+]i in P4H-TM KO astrocytes versus WT in EGTA-containing buffer, n = 16 cells per genotype, 3 mice per genotype. C, qRT–PCR analysis of mRNA expression levels for the translocon complex subunits SEC61 A, B, and G in P4H-TM KO and WT astrocytes, n = 7–12, 3–4 individual cultures per genotype with one to three technical replicates each, 9–12 mice per genotype. D, E, Western blot analysis of expression of translocon subunits SEC61 A, B, and G, in P4H-TM KO and WT astrocytes. Representative Western blots (D) and their quantification, n = 4 individual cultures per genotype, 4 mice per genotype (E) are shown. β-Actin represents a loading control. F, G, Western blot analysis of phosphorylation of elF2a and Erk1/2 and p38 kinases in P4H-TM KO and WT astrocytes after treatment with 10 μm TG for the indicated time points. Representative Western blots (F) of the phospho and total forms are shown. β-Actin is shown to confirm equal protein amount in the samples. Western blot quantification, n = 3–4 individual cultures per genotype, 3–4 mice per genotype (E) are shown. Data information: data are presented as mean ± SEM in A–C, E. In A: *p < 0.05, **p < 0.01 by Holm test after one-way ANOVA for multiple comparisons. In B, C, E, G: *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. r.u., relative unit.